Activation of Protein Kinase D by Signaling through the α Subunit of the Heterotrimeric G Protein Gq *

Protein kinase D (PKD/PKCμ) immunoprecipitated from COS-7 cells transiently transfected with a constitutively active α subunit of Gq (GαqQ209L) exhibited a marked increase in basal activity, which was not further enhanced by treatment of the cells with phorbol 12,13-dibutyrate. In contrast, transient transfection of COS-7 cells with activated Gα12Q229L or Gα13Q226L neither promoted PKD activation nor interfered with the increase of PKD activity induced by phorbol 12,13-dibutyrate. The addition of aluminum fluoride to cells co-transfected with PKD and wild type Gαq induced a marked increase in PKD activity, which was comparable with that induced by expression of GαqQ209L. Treatment with the protein kinase C inhibitor GF I or Ro 31-8220 prevented the increase in PKD activity induced by aluminum fluoride. Expression of a COOH-terminal fragment of Gαq that acts in a dominant negative fashion attenuated PKD activation in response to agonist stimulation of bombesin receptor. PKD activation in response to either Gαq or bombesin was completely prevented by mutation of Ser744 and Ser748 to Ala in the kinase activation loop of PKD. Our results show that Gαqactivation is sufficient to stimulate sustained PKD activation via protein kinase C and indicate that the endogenous Gαqmediates PKD activation in response to acute bombesin receptor stimulation.

structural, enzymological and regulatory properties distinct from other members of the PKC family (8). For example, the catalytic domain of PKD is distantly related to Ca 2ϩ -regulated kinases, and the regulatory region of this kinase contains a putative trans-membrane domain, contains a pleckstrin homology domain that regulates enzyme activity (9,10), and lacks a sequence with homology to a typical PKC autoinhibitory pseudosubstrate motif (6,7). In particular, PKD is rapidly activated in intact cells through a mechanism that involves phosphorylation (8). Exposure of intact cells to phorbol esters, cell-permeant DAGs, or bryostatin induces rapid PKD phosphorylation and activation, which is maintained during cell lysis and immunoprecipitation (9,(11)(12)(13)(14). Several lines of evidence generated by using PKC-specific inhibitors and co-transfection of PKD with constitutively active PKC mutants suggest that PKD is activated by phosphorylation through a novel PKC-dependent signal transduction pathway in vivo (10 -13). The residues Ser 744 and Ser 748 in the activation loop of PKD have been identified as critical phosphorylation sites in PKD activation induced by phorbol esters (15,16).
Recently, we reported that PKD is rapidly activated by a variety of neuropeptide agonists, including bombesin, bradykinin, endothelin, and vasopressin, that signal through heptahelical receptors coupled to heterotrimeric G proteins (13,14,17). Although each of these receptors couples to G q (18) and thereby to phospholipase C (PLC) (19), these data do not define G q as a mediator of PKD activation, because these receptors also couple to other heterotrimeric G proteins including members of the G 12 family that have been recently implicated in pathways leading to PKC activation (20 -23). In order to clarify the G protein pathways leading to PKD activation, we examined whether G␣ q -mediated signaling is sufficient to promote PKD activation in intact cells and whether endogenous G␣ q mediates PKD activation in response to bombesin receptor stimulation.
The results presented here demonstrate that either mutationally activated or aluminum fluoride-stimulated G␣ q induces striking PKD activation through a PKC-dependent pathway. Expression of a COOH-terminal fragment of G␣ q that acts in a dominant negative fashion attenuated PKD activation in response to agonist stimulation of bombesin receptor. PKD activation in response to either G␣ q or bombesin is completely prevented by mutation of Ser 744 and Ser 748 to Ala in the kinase activation loop of PKD. Our results indicate that G␣ q activation is sufficient to stimulate sustained PKD activation via PKC and show that the endogenous G␣ q mediates PKD activation in response to acute bombesin receptor stimulation.

EXPERIMENTAL PROCEDURES
Cell Culture and Transfections-COS-7 cells were maintained by subculture in 10-cm tissue culture plates every 3-4 days in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere containing 10% CO 2 . For experimental dishes, cells were subcultured at 6 ϫ 10 4 cells/ml in 6-cm (5-ml) or 10-cm (10-ml) dishes on the day prior to transfections. All transfections and cotransfections were carried out with equivalent amounts of DNA (6 g/6-cm dish, 12 g/10-cm dish). Transfections were carried out in Opti-MEM (Life Technologies, Inc.) using Lipofectin (Life Technologies) at 10 l/6-cm dish or 20 l/10-cm dish, added to cells in a final volume of 2.5 ml/6-cm dish or 5 ml/10-cm dish, following formation of DNA-Lipofectin complexes according to the protocol provided by the manufacturer. Cells were allowed to take up complexes in the absence of fetal bovine serum for 5-6 h or overnight, and then fetal bovine serum (10% final concentration) in Opti-MEM was added to the dishes to yield a final volume of 5 ml/6-cm dish or 10 ml/10-cm dish. Cells were used for experiments after a further 48 -72 h of incubation.
Polymerase chain reaction was used to generate DNA encoding for the carboxyl-terminal region of G␣ q (residues 305-359) using the murine G␣ q cDNA as a template with sense (5Ј-GCTCAAGCTTCGGCTC-GAGAATTCATCCTGAAAATG-3Ј) and antisense (5Ј-GGTGGATCCT-TAGACCAGATTGTACTCCTTCAG-3Ј) primers. The resulting DNA fragment was subcloned into the BamHI and HindIII restriction sites of pcDNA-3. The fidelity of the polymerase chain reaction was confirmed by DNA sequencing. The BamHI/HindIII DNA fragment was cloned into p⑀GFP-C1 (CLONTECH, Inc., La Jolla, CA) such that the resulting fusion protein produced by this plasmid would be a hybrid ⑀GFP containing G␣ q (residues 305-359) at its carboxyl terminus.
Immunoprecipitations-Transfected COS-7 cells were washed twice with Dulbecco's modified Eagle's medium and equilibrated in 5 ml of the same medium at 37°C for 1-2 h. Some dishes were treated with various pharmacological agents during this equilibration period or with agonists for 10 min at the end of this period, as indicated in the corresponding figure legends. Cells were lysed in buffer A (50 mM Tris-HCl, pH 7.6, 2 mM EGTA, 2 mM EDTA, 1 mM dithiothreitol, 100 g/ml leupeptin, 1 mM 4-(2-aminoethyl)-bengenesulfonyl fluoride, hydrochloride (Pefabloc), and 1% Triton X-100). PKD was immunoprecipitated at 4°C for 3 h with the PA-1 antiserum (1:50 dilution) raised against the synthetic peptide EEREMKALSERVSIL that corresponds to the COOH-terminal region of PKD as described previously (6,26). The immune complexes were recovered using protein A coupled to agarose.
In Vitro Kinase Assays-Immune complexes were washed twice with lysis buffer and then twice with kinase buffer consisting of 30 mM Tris-HCl, pH 7.4, 10 mM MgCl 2 , 1 mM dithiothreitol. Autophosphorylation reactions were initiated by combining 20 l of immune complexes with 5 l of a phosphorylation mixture containing 100 M [␥-32 P]ATP (specific activity, 400 -600 cpm/pmol) in kinase buffer. Following incubation at 30°C for 10 min, the reactions were terminated by the addition of 1 ml of ice cold kinase buffer and placed on ice. Immune complexes were recovered by centrifugation, and the proteins were extracted for SDS-PAGE analysis by the addition of 2ϫ SDS-PAGE sample buffer (200 mM Tris/HCl, pH 6.8, 6% SDS, 2 mM EDTA, 4% 2-mercaptoethanol, 10% glycerol). Dried SDS-PAGE gels were subjected to autoradiography to visualize radiolabeled protein bands.
For assays of exogenous substrate phosphorylation, immune complexes were processed as for autophosphorylation reactions, and then substrate (syntide-2; final concentration 2.5 mg/ml) was added in the presence of 100 M [␥-32 P]ATP (400 -600 cpm/pmol) in kinase buffer (final reaction volume, 30 l). After incubation at 30°C for 10 min, the reactions were terminated by adding 100 l of 75 mM H 3 PO 4 , and 75 l of the mixed supernatant was spotted to Whatman P-81 phosphocellulose paper. Papers were washed thoroughly in 75 mM H 3 PO 4 and dried, and radioactivity incorporated into syntide-2 was determined by detection of Cerenkov radiation in a scintillation counter.
Western Blot Analysis-Samples of cell lysates were directly solubilized by boiling in SDS-PAGE sample buffer. Following SDS-PAGE on 8% gels (for PKD) or 10% gels (for G proteins), proteins were transferred to Immobilon-P membranes (Millipore Corp.), as described previously (11,27) and blocked by overnight incubation with 5% nonfat dried milk in phosphate-buffered saline, pH 7.2. Membranes were incubated at room temperature for 3 h with antisera specifically recognizing either PKD, the different G proteins (G␣ q , G␣ 12 , or G␣ 13 ), or GFP at a 1:250 -1:500 dilution in phosphate-buffered saline containing 3% nonfat dried milk. Immunoreactive bands were visualized using either horseradish peroxidase-conjugated anti-rabbit IgG and subsequent enhanced chemiluminescence detection or 125 I-labeled protein A followed by autoradiography. The G␣ q antiserum was raised against a synthetic peptide corresponding to the COOH-terminal decapeptide of this G protein, which was cross-linked to keyhole limpet hemocyanin with glutaraldehyde. The G␣ 13 antiserum was raised against the synthetic peptide CLHDNLKQLMLQ (which corresponds to the carboxyl-terminal peptide 367-377 of murine G␣ 13 with an NH 2 -terminal cysteine added for coupling) cross-linked to keyhole limpet hemocyanin with the heterobifunctional reagent sulfosuccinimidyl 4-(p-maleimidophenyl) butyrate, as described (28). The antibody for GFP was raised in rabbits to a GST-GFP fusion protein, as recently described (29).
Materials-[␥-32 P]ATP (370 MBq/ml), 32 P i (10 mCi/ml), 125 I-labeled protein A (15 mCi/ml), horseradish peroxidase-conjugated donkey antirabbit Ig, enhanced chemiluminescence reagents, and glutathione-Sepharose were from Amersham Pharmacia Biotech. Protein A-agarose and Pefabloc were from Roche Molecular Biochemicals. Rabbit anti-G␣ 12 was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Opti-MEM and Lipofectin were from Life Technologies. GF I was obtained from Sigma. Ro 31-8220 was from Calbiochem. All other reagents were from standard suppliers or as described and were the highest grade commercially available.

G␣ q QL Induces PKD Activation in COS-7 Cells-Mutations
in the catalytic domain of G␣ subunits that inhibit their intrinsic GTPase activity are known to convert these proteins into constitutively active ␣ subunits (30). To examine the effects of G␣ subunits on PKD activation, COS-7 cells were transiently co-transfected with expression plasmids encoding wild type PKD and constitutively active G␣ mutants G␣ q Q209L, G␣ 12 Q229L, and G␣ 13 Q226L, which are deficient in GTPase activity (24,25,31). PKD was immunoprecipitated from the lysates of transfected cells, and the immune complexes were incubated with [␥-32 P]ATP, subjected to SDS-PAGE, and analyzed by autoradiography to detect the prominent 110-kDa band corresponding to autophosphorylated PKD.
As shown in Fig. 1A, PKD isolated from unstimulated COS-7 cells had low catalytic activity that was markedly activated by PDB stimulation of intact cells (ϳ10-fold increase). In contrast, PKD immunoprecipitated from COS-7 cells overexpressing constitutively active mutant G␣ q QL exhibited a marked increase in basal activity, which was not further enhanced by treatment of the cells with PDB. Transient transfection of COS-7 cells with activated G␣ 12 QL or G␣ 13 QL expression plasmids neither promoted PKD activation nor interfered with the increase of PKD activity induced by PDB. Similarly, overexpression of wild-type G␣ q (Fig. 2) or G␣ 12 and G␣ 13 (results not shown) in COS-7 cells did not induce PKD activation. Western blot analysis confirmed that the cells transfected with the G␣ q QL, G␣ 12 QL, or G␣ 13 QL expression plasmids overexpressed these G␣ subunits and verified the expression of PKD under all these conditions (Fig. 1B). These results suggest that PKD activation in response to G␣ q QL expression is specific for the activated state of this G␣ subunit.
Aluminum Fluoride Stimulates PKD Activation in COS-7 Cells Transfected with Wild Type G␣ q -An increase in PKD activity in response to expression of constitutive active G␣ q could be mediated by mechanisms arising from long term activation of G protein-regulated pathways, e.g. secreted factors that activate cellular receptors in an autocrine manner or alteration in the levels of regulators of G protein function. To assess this possibility, we examined G protein signaling in an acutely regulated system.
Aluminum fluoride activates heterotrimeric G proteins due to its ability to mimic the ␥-phosphoryl group of GTP when complexed with the GDP-bound ␣ subunit (32). We transiently transfected COS-7 cells with vector or wild type G␣ q (rather than the constitutively active form) and then stimulated the cells with either 10 M aluminum fluoride or PDB, as a positive control. PKD activity in immunocomplexes was determined by autophosphorylation or by phosphorylation of syntide-2 (33,34), a synthetic peptide previously demonstrated to be an excellent substrate for PKD (6). As shown in Fig. 2, the addition of aluminum fluoride to cells co-transfected with PKD and wild type G␣ q induced a marked increase in PKD activity, which was comparable with that induced by expression of G␣ q QL. Western blot analysis confirmed that the cells transfected with the G␣ q expression plasmid overexpressed this G␣ subunit ( Fig. 2A). In contrast, the addition of aluminum fluoride to cells transfected with PKD in the absence of G␣ q failed to induce any significant increase in PKD activity. Thus, acute stimulation of G␣ q by aluminum fluoride substantiated the conclusion that G␣ q activation leads to PKD activation.
To verify that the kinase activity induced by either expression of constitutively activated G␣ q or by treatment with aluminum fluoride of cells transfected with wild type G␣ q was due to PKD rather than to the presence of a co-precipitating protein kinase, we examined G␣ q -induced PKD activation in cells transfected with wild type PKD or with a kinase-deficient PKD mutant (PKD K618M) in which lysine 618 in the ATP binding site is substituted by methionine (11). Fig. 3 shows that expression of constitutively activated G␣ q or treatment with aluminum fluoride did not induce detectable kinase activity when COS-7 cells were transfected with PKD K618M, as judged by autophosphorylation or by syntide-2 phosphorylation assays. Western blot analysis verified that the expression levels of wild type PKD and PKD K618M were similar and illustrated that stimulation with aluminum fluoride or expression of constitutively activated G␣ q induced a mobility shift of wild type (but not kinase-deficient) PKD (Fig. 3A). These results demonstrate that the G␣ q -induced kinase activity measured in PKD immunoprecipitates is due to the activation of PKD rather than to co-immunoprecipitating kinases.

Aluminum Fluoride Stimulates PKD Phosphorylation in COS-7 Cells Transfected with Wild Type G␣ q -
The preceding experiments demonstrated that mutationally activated or aluminum fluoride-stimulated G␣ q increased PKD autophosphorylation in in vitro kinase assays. We next examined whether G␣ q activation induces PKD phosphorylation in intact cells. COS-7 cells transfected with PKD, PKD, and G␣ q or vectors (pcDNA3 and pcDNA1, as indicated in Fig. 4) were metabolically labeled with 32 P i and then stimulated with 10 M aluminum fluoride or 200 nM PDB. Cells were lysed, and PKD was immunoprecipitated with PA-1 antiserum and analyzed by SDS-PAGE and autoradiography. As shown in Fig. 4, aluminum fluoride induced PKD phosphorylation in COS-7 cells transfected with PKD and G␣ q but did not produce any detectable effect in cells transfected with PKD alone. As a control, we verified in parallel cultures that PDB induced PKD phosphorylation in cells transfected with PKD either with or without G␣ q . These results indicate that stimulation of G␣ q by aluminum fluoride induces PKD phosphorylation in intact cells.
The PKC Inhibitors GF I and Ro 31-8220 Prevent PKD Activation by Aluminum Fluoride in COS-7 Cells Transfected with G␣ q -Next, we determined whether PKCs mediate PKD activation induced by G␣ q activation using inhibitors that discriminate between PKCs and PKD (11,13). COS-7 cells transiently co-transfected with wild type G␣ q and PKD were treated for 1 h with the PKC inhibitors GF I (also known as GF 109203X or bisindolylmaleimide I) and Ro 31-8220 (35,36) prior to stimulation with 10 M aluminum fluoride or PDB. Treatment with either GF I or Ro 31-8220 prevented the increase in PKD activity induced by aluminum fluoride in G␣ q -transfected COS-7 cells, as shown by autophosphorylation (Fig. 5A) or syntide-2 phosphorylation assays (Fig. 5B). In contrast, GFV, which is structurally related to GF I but biologically inactive, did not affect PKD activation in response to either aluminum fluoride or PDB. Previously, we demonstrated that GF I and Ro 31-8220 do not directly inhibit PKD activity when added to the in vitro kinase assay at concentrations identical to those required to block PKD activation by aluminum fluoride in G␣ qtransfected COS-7 cells (11,13). Thus, the results shown in Fig.  5 imply that G␣ q -mediated PKD activation in intact COS-7 cells is mediated by PKC.
Substitution of Ser 744 and Ser 748 by Alanine Prevents PKD Activation in Response to G␣ q -A critical aspect in the regulation of protein kinases that function in signaling cascades is the phosphorylation of activating residues located in a region spanning the highly conserved sequences DFG (in kinase subdomain VII) and APE (in kinase subdomain VIII) of the kinase catalytic domain termed the "activation loop" or "activation segment" (37,38). Recently, we identified Ser 744 and Ser 748 as activating residues in the activation loop of PKD and demonstrated that these residues are phosphorylated in intact cells in response to PDB stimulation (15). Here, we examined whether these residues are also important in PKD activation in response to G␣ q activation or bombesin receptor stimulation.
If Ser 744 and Ser 748 are critical target sites for activating phosphorylation(s) events in response to G␣ q , their conversion to alanine should reduce or eliminate G␣ q -mediated activation of PKD. To test this possibility, we used PKD mutants with single or double substitutions of these residues cloned in the expression vector pcDNA3 (i.e. PKD-S744A, PKD-S748A, or

FIG. 4. Aluminum fluoride stimulates PKD phosphorylation in COS-7 cells transfected with wild type G␣ q protein.
Exponentially growing COS-7 cells were co-transfected with pcDNA3-PKD (PKD) and pcDNA1 or pcDNA1 encoding wild type G␣ q (␣qwt). The control cells were transfected with pcDNA3 and pcDNA1. Three days after transfection, cells were washed twice with P i -free Dulbecco's modified Eagle's medium, incubated in this medium for 30 min, and metabolically labeled with carrier-free 32  PKD-S744A/S748A). COS-7 cells, co-transfected with wild type PKD or PKD mutants and either G␣ q QL (Fig. 6A, upper panel) or G␣ q (Fig. 6A, lower panel), were treated with or without aluminum fluoride or PDB and lysed. PKD was immunoprecipitated from the extracts with the PA-1 antibody. The immunocomplexes were incubated with [␥-32 P]ATP and analyzed by SDS-PAGE and autoradiography to determine the level of PKD activity by autophosphorylation.
As shown in Fig. 6, PKD isolated from unstimulated cells transfected with G␣ q had low catalytic activity that was markedly activated to the same degree by G␣ q QL, aluminum fluoride-stimulated G␣ q , or PDB. Substitution of both Ser 744 and Ser 748 for Ala in PKD completely blocked kinase activation induced by either mutationally activated or aluminum fluoridestimulated G␣ q . Single substitutions of either Ser 744 or Ser 748 for Ala resulted in PKD mutants that displayed reduced activity after stimulation (ϳ50% decrease in both single Ala mutants compared with stimulated wild type PKD). In all cases, the protein expression levels of the transfected PKD mutants were comparable with that of wild type PKD, as shown by Western blot analysis (Fig. 6B). Thus, alanine substitution of Ser 744 and Ser 748 in the activation loop of PKD prevents the activation of this enzyme by G␣ q in vivo.
Some protein kinases that are activated by phosphorylation in the activation loop can be rendered constitutively active by substitution of the phosphorylated residue(s) for glutamic acid (39). As shown in Fig. 6A and in agreement with recent results (15), replacement of both serine residues with glutamic acid (PKD-S744E/S748E) markedly increased basal activity. Interestingly, the activity of the PKD-S744E/S748E mutant was not further increased by mutationally activated G␣ q , aluminum fluoride-stimulated G␣ q , or PDB, suggesting that phosphorylation of these two sites induces maximal PKD activation in response to these pathways. Western blot analysis verified that the expression levels of wild type PKD and constitutive activated PKD mutants were similar (Fig. 6B).
Substitution of Ser 744 and Ser 748 by Alanine Prevents PKD Activation in Response to Bombesin-Bombesin and its mammalian counterpart gastrin-releasing peptide bind to a heptahelical receptor (40,41) that couples to G␣ q with high affinity (42,43) and induces a complex array of early signaling events (44). Previously, we demonstrated that bombesin induces a rapid increase in PKD activity in Swiss 3T3 cells (13). Here, we examined whether bombesin-induced PKD activation requires the phosphorylation of Ser 744 /Ser 748 in the activation loop. COS-7 cells transiently co-transfected with bombesin receptor and wild type PKD or PKD-S744A, PKD-S748A, or PKD-S744A/S748A were treated with or without bombesin or PDB for 10 min and then lysed. PKD activity in the immune complexes was measured by autophosphorylation. As shown in prevents the activation of this enzyme by either bombesin, G␣ q , or PDB in vivo.
The results presented in Fig. 7 also show that the high constitutive kinase activity of the PKD-S744E/S748E mutant was not significantly further stimulated by bombesin, suggesting that phosphorylation of these two sites induces maximal PKD activation in response to this neuropeptide. The lack of either basal activity or bombesin-induced activation in PKD immunoprecipitates from COS-7 cells transfected with either PKD or PKD-S744E/S748E carrying the kinase-inactivating D733A mutation (45) indicates that the kinase activity measured is due to the activation of PKD rather than to co-immunoprecipitating protein kinases (Fig. 7A). In all cases, the protein expression levels of the transfected PKD mutants were comparable with that of wild type PKD, as shown by Western blot analysis (Fig. 7B).
Role of Endogenous G␣ q in Mediating PKD Activation in Response to Bombesin Receptor Activation-The COOH terminus of G proteins plays a key role in their interaction with cognate receptors (46). Recently, peptides corresponding to this region of G␣ q or G␣ i have been shown to target the receptor-G protein interface in a selective manner and thereby block receptor-mediated PLC activation (47) and inwardly rectifying K ϩ channel activity (48,49), respectively. For example, transient transfection of COS-7 cells with ␣ 1B -adrenergic receptors or M 1 muscarinic receptors and the COOH-terminal region of G␣ q attenuated inositol phosphate production in response to receptor activation (47).
In the present study, a dominant negative strategy was also used to test the role of endogenous G␣ q in bombesin receptormediated PKD activation. We generated chimeric fusion proteins between the COOH-terminal region of G␣ q (referred to as G␣ q CT) and GFP from Aequorea victoria, which forms an independent 30-kDa domain with inherent fluorescence (50). Initially, we verified that the GFP-G␣ q CT chimera is expressed in transiently transfected COS-7 cells as judged by Western blot analysis using antibodies directed against either GFP or the COOH-terminal region of G␣ q (Fig. 8A). In addition, we also visualized the expression of the GFP-G␣ q CT chimera by examining GFP fluorescence in individual COS-7 cells (results not shown). Next, we determined whether expression of GFP-G␣ q CT interferes with PKD activation via the bombesin receptor. COS-7 cells were co-transfected with PKD, bombesin receptor, and either GFP-G␣ q CT or GFP. After 72 h, the cells were challenged with either bombesin or PDB for 10 min and then lysed. PKD activity, after immunoprecipitation, was assayed by autophosphorylation or syntide-2 phosphorylation. The results illustrated in Fig. 8 (B and C) demonstrate that expression of GFP-G␣ q CT markedly attenuated the increase of PKD activity induced by bombesin. In contrast, expression of GFP-G␣ q CT did not interfere with PKD activation in response to PDB, which directly stimulates PKC leading to PKD activation and therefore bypasses the receptor/G␣ q interaction. These results indicate that endogenous G␣ q mediates PKD activation in response to bombesin receptor activation.
Conclusion-PKD/PKC is a serine/threonine protein kinase with distinct structural, enzymological, and regulatory properties. Recently, activation of a number of receptors that couple to heterotrimeric G proteins, including those for bombesin, bradykinin, endothelin, and vasopressin, has been shown to stimulate PKD activation in a variety of cell types. Here, we examined the mechanism(s) by which G protein-coupled receptors lead to PKD activation.
It is generally thought that G q stimulation of the ␤ isoforms of PLC catalyzes the production of inositol 1,4,5-trisphosphate that triggers the release of Ca 2ϩ from internal stores and DAG that directly activates the classic and novel isoforms of PKC (reviewed in Ref. 19). Accordingly, it is well established that constitutively activated forms of G␣ q stimulate the ␤ isoforms of PLC in vitro and induce persistent stimulation of inositol phosphate production in intact cells (19). In contrast, the other important arm of this bifurcating signaling pathway, namely the production of DAG and the activation of PKC, has been less frequently measured. In this context, it is relevant that DAG, unlike inositol 1,4,5-trisphosphate, can be generated through routes other than phosphoinositide hydrolysis mediated by G␣ q -stimulated PLC (51) and that G q -coupled receptors also interact with other heterotrimeric G proteins including members of the G 12 family that have been recently implicated in pathways leading to PKC activation (20 -22). It is also of interest that expression of active G␣ q did not induce persistent activation of mitogen-activated protein kinases in either NIH 3T3 cells (52) or PC12 cells (53), suggesting that chronic G␣ q -PLC activation could lead to PKC down-regulation. Consequently, we examined whether G␣ q -mediated signaling is sufficient to promote PKD activation in intact cells and whether endogenous G␣ q mediates PKD activation in response to bombesin receptor stimulation.
Our results demonstrate that either mutationally activated or aluminum fluoride-stimulated G␣ q induces striking PKD activation through a PKC-dependent pathway. PKD activation in response to bombesin receptor stimulation, G␣ q , or PDB is completely prevented by mutation of Ser 744 and Ser 748 to Ala in the kinase activation loop of PKD. Furthermore, none of these stimuli induced a further increase in PKD activity when Ser 744 and Ser 748 were mutated to Glu to mimic the phosphorylated residues. These data indicate that bombesin receptor activation, G␣ q stimulation, and PDB lead to PKD activation through the same mechanism, namely phosphorylation of Ser 744 /Ser 748 in the activation loop of PKD.
Dominant negative strategies to uncouple heptahelical receptors from their cognate G proteins have received much attention, but only recently has it been shown that expression of the COOH-terminal region of G proteins can competitively inhibit receptor-G protein interaction (47,49). For example, expression of the last 55 amino acids of G␣ q has been shown to target the receptor-G q interface in a selective manner and thereby block receptor-mediated PLC activation in cultured cells and in transgenic mice (47). Here, we pursued a similar strategy and demonstrate, for the first time, that expression of a chimeric fusion protein consisting of the COOH-terminal region of G␣ q and GFP attenuated PKD activation in response to agonist stimulation of bombesin receptor but not in response to PDB, which bypasses the receptor. GFP conjugates with COOH-terminal peptides of G proteins may provide a useful approach to monitor the expression of competing (dominant negative) G protein peptides in intact cells.
Expression of constitutively active G␣ q is known to induce a variety of biological responses including transformation (54,55), differentiation (55), and apoptosis (56). G q signaling is of great interest in the development and decompensation of cardiac hypertrophy (57)(58)(59). Consequently, there is a renewed interest in identifying downstream targets that are persistently activated by expression of activated G␣ q (60). Our results indicate that G␣ q activation is sufficient to stimulate sustained PKD activation via PKC and show that the endogenous G␣ q mediates PKD activation in response to acute bombesin receptor stimulation.